Methods of inhibiting VEGF-C

ABSTRACT

The present invention provides RNA molecules (e.g., antisense, RNAi, or siRNA) specific for VEGF-C, and further provides methods of reducing expression of VEGF-C in cells (e.g., cancer cells).

CLAIM OF PRIORITY

This patent application is a Divisional Application of U.S. applicationSer. No. 11/194,276 filed on Aug. 1, 2005, which claims priority to U.S.application Ser. No. 60/598,003 filed on Aug. 2, 2004. The instantapplication claims the benefit of the listed applications, which arehereby incorporated by reference herein in their entirety.

TECHNICAL FIELD

This invention relates to siRNA molecules, and more particularly toVEGF-C siRNA molecules.

BACKGROUND

Vascular endothelial growth factor C (VEGF-C; also known as vascularpermeability factor) is a member of the signaling growth factor family.VEGF-C is a growth factor active in angiogenesis and endothelial cellgrowth, and stimulates their proliferation and migration. VEGF-C alsohas effects on the permeability of blood vessels. VEGF-C may function inangiogenesis of the venous and lymphatic vascular systems duringembryogenesis, and also in the maintenance of differentiated lymphaticendothelium in adults. VEGF-C generally has several cysteine-richmotifs, and usually is about 47 kDa. VEGF-C is expressed in the spleen,lymph node, thymus, appendix, bone marrow, heart, placenta, ovary,skeletal muscle, prostate, testis, colon and small intestine, fetalliver, fetal lung, and fetal kidney.

SUMMARY

The present invention provides RNA molecules (e.g., antisense, RNAi, orsiRNA) specific for VEGF-C, and further provides methods of reducingexpression of VEGF-C in a cell (e.g., a cancer cell).

In one aspect, the invention provides an isolated nucleic acid moleculehaving a first portion. Generally, the first portion is no more than 30nucleotides in length, and includes the sequence 5′-AAG ATC TGG AGG AGCAGT TAC-3′ (SEQ ID NO: 1), 5′AAA GGA GGC TGG CAA CAT AAC-3′ (SEQ IDNO:2), 5′-AAC CTC CAT GTT GTG TCC GTC-3′ (SEQ ID NO:3), 5′-AAG ACC TGCCCC ACC AAT TAC-3′ (SEQ ID NO:4), or 5′-AAG AAG TGT GTC GTT GTG TCC-3′(SEQ ID NO:5).

In another aspect, the invention provides methods of reducing theexpression of VEGF-C in a cell. Such methods include introducing anisolated nucleic acid molecule in to the cell in an amount sufficient toreduce the expression of VEGF-C. Generally, the nucleic acid moleculehas a first portion that is no more than 30 nucleotides in length. Thefirst portion typically includes the sequence 5′-AAG ATC TGG AGG AGC AGTTAC-3′ (SEQ ID NO:1), 5′-AAA GGA GGC TGG CAA CAT AAC-3′ (SEQ ID NO:2),5′-AAC CTC CAT GTT GTG TCC GTC-3′ (SEQ ID NO:3), 5′-AAG ACC TGC CCC ACCAAT TAC-3′ (SEQ ID NO:4), or 5′-AAG AAG TGT GTC GTT GTG TCC-3′ (SEQ IDNO:5). According to the invention, the nucleic acid molecule reducesexpression of VEGF-C in the cell. VEGF-C expression can be reduced by atleast 10%.

The nucleic acid molecules described above also can include a secondportion having a sequence complementary to the first portion. Suchnucleic acid molecules further can include a linking sequence that joinsthe first portion and the second portion. For example, the linkingsequence can form a loop of a hairpin. Typically, the linking sequenceis about 4 to about 10 nucleotides in length, and the first portion isfrom 19 to 23 nucleotides in length. The invention also provides forvectors containing such nucleic acid molecules, as well as host cellscontaining such vectors.

A representative cell in which VEGF-C expression can be reduced is acancer cell. Such cancer cells can be epithelially-derived, and caninclude, for example, a head and neck cancer cell, a breast cancer cell,a colon cancer cell, and a prostate cancer cell. In various embodiments,the cancer cell is in vivo; the cancer cell is in a mammal (e.g., ahuman). It is a feature of the invention that proliferation of thecancer cell is inhibited.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. In addition, the materials, methods, andexamples are illustrative only and not intended to be limiting. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedrawings and detailed description, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is a graph showing the results of quantitative RT-PCR of VEGF-Cexpression in head and neck cancer cells transfected with a VEGF-CsiRNA.

Like reference symbols in the various drawings indicate like elements.

DETAILED DESCRIPTION

Several RNA molecules have been identified that are specific for VEGF-Cand that can selectively reduce expression of VEGF-C in a cell. Theinvention provides for such VEGF-C RNA molecules, the DNA moleculesencoding such RNA molecules, and also provides for methods of using thenucleic acid molecules of the invention to reduce the expression ofVEGF-C. The RNA molecules of the invention can be used in a number ofdifferent forms including antisense, RNAi, and siRNA. Although thefollowing discussion focuses on siRNA, the methods of the invention arenot limited by a particular mechanism.

VEGF-C siRNA Molecules

A “small interfering RNA” or “short interfering RNA” or “siRNA” or“short hairpin RNA” or “shRNA” is a double-stranded RNA molecule that iscomplementary to a target nucleic acid sequence, for example, VEGF-C. Adouble-stranded RNA molecule is formed by the complementary pairingbetween a first RNA portion and a second RNA portion. The length of eachportion generally is less than 30 nucleotides in length (e.g., 29, 28,27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, or10 nucleotides). In some embodiments, the length of each portion is 19to 25 nucleotides in length. In some siRNA molecules, the complementaryfirst and second portions of the RNA molecule are the “stem” of ahairpin structure. The two portions can be joined by a linking sequence,which can form the “loop” in the hairpin structure. The linking sequencecan vary in length. In some embodiments, the linking sequence can be 5,6, 7, 8, 9, 10, 11, 12 or 13 nucleotides in length. A representativelinking sequence is 5′-TTC AGA AGG-3′, but any of a number of sequencescan be used to join the first and second portions. The first and secondportions are complementary but may not be completely symmetrical, as thehairpin structure may contain 3′ or 5′ overhang nucleotides (e.g., a 1,2, 3, 4, or 5 nucleotide overhang).

RNA molecules have been shown by many researchers to be effective insuppressing mRNA accumulation. siRNA-mediated suppression of nucleicacid expression is specific as even a single base pair mismatch betweensiRNA and the targeted nucleic acid can abolish the action of RNAinterference. siRNAs generally do not elicit anti-viral responses.

There are well-established criteria for designing siRNAs (see, e.g.,Elbashire et al., 2001, Nature, 411:494-8; Amarzguioui et al., 2004,Biochem. Biophys. Res. Commun., 316(4):1050-8; Reynolds et al., 2004,Nat. Biotech., 22(3):326-30). Details can be found in the websites ofseveral commercial vendors such as Ambion, Dharmacon, GenScript, andOligoEngine. The sequence of any potential siRNA candidate generally ischecked for any possible matches to other nucleic acid sequences orpolymorphisms of nucleic acid sequence using the BLAST alignment program(see ncbi.nlm.nih.gov on the World Wide Web). Typically, a number ofsiRNAs have to be generated and screened in order to compare theireffectiveness.

Once designed, the siRNAs of the present invention can be generated byany method known in the art, for example, by in vitro transcription,recombinantly, or by synthetic means (e.g., having either a TT or a UUoverhang at the 3′ end). siRNAs can be generated in vitro by using arecombinant enzyme, such as T7 RNA polymerase, and DNA oligonucleotidetemplates, or can be prepared in vivo, for example, in cultured cells(see, for example, Elbashir et al., supra; Brummelkamp et al., supra;and Lee et al., 2002, Nat. Biotech., 20:500-5).

In addition, strategies have been described for producing a hairpinsiRNA from vectors containing a RNA polymerase III promoter. Variousvectors have been constructed for generating hairpin siRNAs in hostcells using either an H1-RNA or an snU6 RNA promoter. A RNA molecule asdescribed above (e.g., a first portion, a linking sequence, and a secondportion) can be operably linked to such a promoter. When transcribed byRNA polymerase III, the first and second portions form a duplexed stemof a hairpin and the linking sequence forms a loop. The pSuper vector(OligoEngines Ltd., Seattle, Wash.) also can be used to generate siRNA.

A TTTTT penta-nucleotide usually is attached to the end of the secondportion (i.e., the antisense strand) in a vector to serve as aterminator for RNA polymerase III transcription. For that reason, siRNAcandidates that contain more than three consecutive Ts should be avoidedsince four or more consecutive Ts in the template nucleic acid triggerstermination of RNA polymerase III transcription.

Several techniques can be used to test the effect of different siRNAconstructs on cellular mRNA and/or protein levels. For example, dual-GFPtransfection, CHO-cell double transfection based on an antibody/epitopespecificity, quantitative RT-PCR, Northern blots, Western blots,immunofluorescence, and Hygro/Neo selection. These methods are wellknown in the art.

VEGF-C Nucleic Acids and Polypeptides

As used herein, the term “nucleic acid molecule” can include DNAmolecules and RNA molecules and analogs of a DNA or RNA moleculegenerated using nucleotide analogs. A nucleic acid molecule of theinvention can be single-stranded or double-stranded, and thestrandedness will depend upon its intended use. Fragments or portions ofthe disclosed nucleotide sequences are also encompassed by the presentinvention. By “fragment” or “portion” is meant less than full length ofthe nucleotide sequence.

The invention further encompasses nucleic acid molecules that differ innucleotide sequence. Nucleic acid molecules that differ in sequence fromthe original nucleic acid sequence can be generated by standardtechniques, such as site-directed mutagenesis, PCR-mediated mutagenesis,or oligonucleotide-mediated mutagenesis. In addition, nucleotide changescan be introduced randomly along all or part of a nucleic acid moleculesuch as by saturation mutagenesis. Alternatively, nucleotide changes canbe introduced into a sequence by chemically synthesizing a nucleic acidmolecule having such changes.

To calculate percent sequence identity, two sequences are aligned andthe number of identical matches of nucleotides or amino acid residuesbetween the two sequences is determined. The number of identical matchesis divided by the length of the aligned region (i.e., the number ofaligned nucleotides or amino acid residues) and multiplied by 100 toarrive at a percent sequence identity value. It will be appreciated thatthe length of the aligned region can be a portion of one or bothsequences up to the full-length size of the shortest sequence. It willbe appreciated that a single sequence can align differently with othersequences and hence, can have different percent sequence identity valuesover each aligned region. It is noted that the percent identity value isusually rounded to the nearest integer. For example, 78.1%, 78.2%,78.3%, and 78.4% are rounded down to 78%, while 78.5%, 78.6%, 78.7%,78.8%, and 78.9% are rounded up to 79%. It is also noted that the lengthof the aligned region is always an integer.

The alignment of two or more sequences to determine percent sequenceidentity is performed using the algorithm described by Altschul et al.(1997, Nucleic Acids Res., 25:3389-402) as incorporated into BLAST(basic local alignment search tool) programs, available atncbi.nlm.nih.gov on the World Wide Web. BLAST searches can be performedto determine percent sequence identity between a nucleic acid moleculeof the invention and any other sequence or portion thereof aligned usingthe Altschul et al. algorithm. BLASTN is the program used to align andcompare the identity between nucleic acid sequences, while BLASTP is theprogram used to align and compare the identity between amino acidsequences. When utilizing BLAST programs to calculate the percentidentity between a sequence of the invention and another sequence, thedefault parameters of the respective programs are used. Sequenceanalysis of nucleic acid sequences can be performed used BLAST version2.2.9 (updated on May 12, 2004).

As used herein, an “isolated” nucleic acid molecule is a nucleic acidmolecule that is separated from other nucleic acid molecules that areusually associated with the isolated nucleic acid molecule. Thus, an“isolated” nucleic acid molecule includes, without limitation, a nucleicacid molecule that is free of sequences that naturally flank one or bothends of the nucleic acid in the genome of the organism from which theisolated nucleic acid is derived (e.g., a cDNA or genomic DNA fragmentproduced by PCR or restriction endonuclease digestion). Such an isolatednucleic acid molecule is generally introduced into a vector (e.g., acloning vector, or an expression vector) for convenience of manipulationor to generate a fusion nucleic acid molecule. In addition, an isolatednucleic acid molecule can include an engineered nucleic acid moleculesuch as a recombinant or a synthetic nucleic acid molecule. A nucleicacid molecule existing among hundreds to millions of other nucleic acidmolecules within, for example, a nucleic acid library (e.g., a cDNA, orgenomic library) or a portion of a gel (e.g., agarose, orpolyacrylamine) containing restriction-digested genomic DNA is not to beconsidered an isolated nucleic acid.

Isolated nucleic acid molecules of the invention can be obtained usingtechniques routine in the art. For example, isolated nucleic acidswithin the scope of the invention can be obtained using any methodincluding, without limitation, recombinant nucleic acid technology,and/or the polymerase chain reaction (PCR). General PCR techniques aredescribed, for example in PCR Primer: A Laboratory Manual, Dieffenbach &Dveksler, Eds., Cold Spring Harbor Laboratory Press, 1995. Recombinantnucleic acid techniques include, for example, restriction enzymedigestion and ligation, which can be used to isolate a nucleic acidmolecule of the invention. Isolated nucleic acids of the invention alsocan be chemically synthesized, either as a single nucleic acid moleculeor as a series of oligonucleotides. In addition, isolated nucleic acidmolecules of the invention also can be obtained by mutagenesis usingcommon molecular cloning techniques (e.g., site-directed mutagenesis).Possible mutations include, without limitation, deletions, insertions,substitutions, and combinations thereof.

Vectors containing nucleic acid molecules also are provided by theinvention. Vectors, including expression vectors, suitable for use inthe present invention are commercially available and/or produced byrecombinant DNA technology methods routine in the art. A vectorcontaining a nucleic acid molecule can have elements necessary forexpression operably linked to such a nucleic acid, and further caninclude sequences such as those encoding a selectable marker (e.g., asequence encoding antibiotic resistance), and/or those that can be usedin purification of a polypeptide (e.g., a His tag). A “vector” isdefined to include any viral vector, as well as any plasmid, cosmid,phage, or binary vector. Vectors can integrate into the cellular genomeor exist extrachromosomally (e.g., an autonomous replicating plasmidwith an origin of replication).

Elements necessary for expression include nucleic acid sequences thatdirect and regulate expression of nucleic acid coding sequences. Oneexample of an element necessary for expression is a promoter sequence.Examples of promoters that may be used in the present invention includethe mouse U6 RNA promoters, synthetic human H1RNA promoters, SV40, CMV,RSV, RNA polymerase II, and RNA polymerase III promoters. Elementsnecessary for expression also can include ribosome-binding sites,introns, enhancer sequences, response elements, or inducible elementsthat modulate expression of a nucleic acid. Elements necessary forexpression can be of bacterial, yeast, insect, mammalian, or viralorigin and vectors can contain a combination of elements from differentorigins. Elements necessary for expression are described, for example,in Goeddel, 1990, Gene Expression Technology: Methods in Enzymology,185, Academic Press, San Diego, Calif. As used herein, operably linkedmeans that a promoter and/or other regulatory element(s) are positionedin a vector relative to a nucleic acid in such a way as to direct orregulate expression of the nucleic acid. A nucleic acid can beoperably-linked to regulatory sequences in sense or antisenseorientation. For example, in the case of siRNA constructs, expressionmay refer to the transcription of the siRNA only. In addition,expression can refer to the transcription of sense mRNA and may alsorefer to the production of protein.

In one embodiment of the present invention, a vector contains an H1-RNApromoter that is operably linked to a nucleic acid sequence encoding asiRNA. Thus, the H1-RNA promoter initiates the transcription of thesiRNA. In another embodiment, the promoter is regulatable, providinginducible expression of the siRNA.

Another aspect of the invention pertains to host cells into which avector of the invention, e.g., an expression vector, or an isolatednucleic acid molecule of the invention has been introduced. The term“host cell” refers not only to the particular cell but also to theprogeny or potential progeny of such a cell. A host cell can be anyprokaryotic or eukaryotic cell. For example, host cells can includebacterial cells such as E. coli, insect cells, yeast cells, or mammaliancells (such as Chinese hamster ovary cells (CHO) or COS cells). Othersuitable host cells are known to those skilled in the art. Many methodsfor introducing nucleic acids into host cells, both in vivo and invitro, are well known to those skilled in the art and include, withoutlimitation, calcium phosphate precipitation, electroporation, heatshock, lipofection, microinjection, and viral-mediated nucleic acidtransfer.

A “polypeptide” refers to a polypeptide encoded by a nucleic acidmolecule. The term “purified” polypeptide as used herein refers to apolypeptide that has been separated or purified from cellular componentsthat naturally accompany it. Typically, the polypeptide is considered“purified” when it is at least 70% (e.g., at least 75%, 80%, 85%, 90%,95%, or 99%) by dry weight, free from the proteins and naturallyoccurring molecules with which it is naturally associated. Since apolypeptide that is chemically synthesized is, by nature, separated fromthe components that naturally accompany it, a synthetic polypeptide is“purified.”

Polypeptides can be purified from natural sources (e.g., a biologicalsample) by known methods such as DEAE ion exchange, gel filtration, andhydroxyapatite chromatography. A purified polypeptide also can beobtained by expressing a nucleic acid in an expression vector, forexample. In addition, a purified polypeptide can be obtained by chemicalsynthesis. The extent of purity of a polypeptide can be measured usingany appropriate method, e.g., column chromatography, polyacrylamide gelelectrophoresis, or HPLC analysis.

In addition to naturally-occurring polypeptides, the skilled artisanwill further appreciate that changes can be introduced into a nucleicacid molecule as discussed herein, thereby leading to changes in theamino acid sequence of the encoded polypeptide. For example, changes canbe introduced into a nucleic acid coding sequence leading toconservative and/or non-conservative amino acid substitutions at one ormore amino acid residues. A “conservative amino acid substitution” isone in which one amino acid residue is replaced with a different aminoacid residue having a similar side chain. Similarity between amino acidresidues has been assessed in the art. For example, Dayhoff et al.(1978, in Atlas of Protein Sequence and Structure, Vol. 5, Suppl. 3, pp345-352) provides frequency tables for amino acid substitutions that canbe employed as a measure of amino acid similarity. A non-conservativesubstitution is one in which an amino acid residue is replaced with anamino acid residue that does not have a similar side chain.

The invention also provides for chimeric or fusion polypeptides. As usedherein, a “chimeric” or “fusion” polypeptide includes one polypeptideoperatively linked to a heterologous polypeptide. The heterologouspolypeptide can be at either the N-terminus or C-terminus of thepolypeptide. Within a chimeric or fusion polypeptide, the term“operatively linked” is intended to indicate that the two polypeptidesare encoded in-frame relative to one another. In a fusion polypeptide,the heterologous polypeptide generally has a desired property such asthe ability to purify the fusion polypeptide (e.g., by affinitypurification). A chimeric or fusion polypeptide of the invention can beproduced by standard recombinant DNA techniques, and can usecommercially available vectors.

A polypeptide commonly used in a fusion polypeptide for purification isglutathione S-transferase (GST), although numerous other polypeptidesare available and can be used. In addition, a proteolytic cleavage sitecan be introduced at the junction between a polypeptide and aheterologous polypeptide to enable separation of the two polypeptidessubsequent to purification of the fusion polypeptide. Enzymes thatcleave such proteolytic sites include Factor Xa, thrombin, orenterokinase. Representative expression vectors encoding a heterologouspolypeptide that can be used in affinity purification of a polypeptideinclude pGEX (Pharmacia Biotech Inc; Smith & Johnson, 1988, Gene,67:31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5(Pharmacia, Piscataway, N.J.).

Detection of Nucleic Acids and Polypeptides

Nucleic acid molecules and polypeptides can be detected using a numberof different methods. Methods for detecting nucleic acids include, forexample, PCR and nucleic acid hybridizations (e.g., Southern blot,Northern blot, or in situ hybridizations). Specifically,oligonucleotides (e.g., oligonucleotide primers) capable of amplifying atarget nucleic acid can be used in a PCR reaction. PCR methods generallyinclude the steps of obtaining a sample, isolating nucleic acid (e.g.,DNA, RNA, or both) from the sample, and contacting the nucleic acid withone or more oligonucleotide primers that hybridize(s) with specificityto the template nucleic acid under conditions such that amplification ofthe template nucleic acid occurs. In the presence of a template nucleicacid, an amplification product is produced. Conditions for amplificationof a nucleic acid and detection of an amplification product are known tothose of skill in the art (see, e.g., PCR Primer: A Laboratory Manual,1995, Dieffenbach & Dveksler, Eds., Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y.; and U.S. Pat. Nos. 4,683,195; 4,683,202;4,800,159; and 4,965,188). Modifications to the original PCR also havebeen developed. For example, anchor PCR, RACE PCR, RT-PCR, or ligationchain reaction (LCR) are additional PCR methods known in the art (see,e.g., Landegran et al., 1988, Science, 241:1077-1080; and Nakazawa etal., 1994, Proc. Natl. Acad. Sci. USA, 91:360-364).

As used herein, “standard amplification conditions” refer to the basiccomponents of an amplification reaction mix, and cycling conditions thatinclude multiple cycles of denaturing the template nucleic acid,annealing the oligonucleotide primers to the template nucleic acid, andextension of the primers by the polymerase to produce an amplificationproduct (see, for example, U.S. Pat. Nos. 4,683,195; 4,683,202;4,800,159; and 4,965,188). The basic components of an amplificationreaction mix generally include, for example, about 10-25 nmole of eachof the four deoxynucleoside triphosphates, (e.g., dATP, dCTP, dTTP, anddGTP, or analogs thereof), 10-100 pmol of each primer, template nucleicacid, and a polymerase enzyme. The reaction components are generallysuspended in a buffered aqueous solution having a pH of between about 7and about 9. The aqueous buffer can further include one or moreco-factors (e.g., Mg²⁺, K⁺) required by the polymerase. Additionalcomponents such as DMSO are optional. Template nucleic acid is typicallydenatured at a temperature of at least about 90° C., and extension fromprimers is typically performed at a temperature of at least about 72° C.

The annealing temperature can be used to control the specificity ofamplification. The temperature at which primers anneal to templatenucleic acid must be below the Tm of each of the primers, but highenough to avoid non-specific annealing of primers to the templatenucleic acid. The Tm is the temperature at which half of the DNAduplexes have separated into single strands, and can be predicted for anoligonucleotide primer using the formula provided in section 11.46 ofSambrook et al. (1989, Molecular Cloning: A Laboratory Manual, 2nd Ed.,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).Non-specific amplification products are detected as bands on a gel thatare not the size expected for the correct amplification product. It canbe appreciated by those of skill in the art that appropriate positiveand negative controls should be performed with every set ofamplification reactions to avoid uncertainties related to contaminationand/or non-specific annealing of oligonucleotide primers and extensiontherefrom.

A pair of primers in an amplification reaction must anneal to oppositestrands of the template nucleic acid, and should be an appropriatedistance from one another such that the polymerase can effectivelypolymerize across the region and such that the amplification product canbe readily detected using, for example, electrophoresis. Oligonucleotideprimers can be designed using, for example, a computer program such asOLIGO (Molecular Biology Insights Inc., Cascade, Colo.) to assist indesigning primers that have similar melting temperatures. Typically,oligonucleotide primers are 10 to 30 or 40 or 50 nucleotides in length(e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, or 50 nucleotides in length), but can be longeror shorter if appropriate amplification conditions are used.Oligonucleotides of the invention can be obtained by restriction enzymedigestion of a nucleic acid molecule or can be prepared by standardchemical synthesis and other known techniques.

Alternatively, a nucleic acid can be detected using a labeledoligonucleotide probe capable of hybridizing to nucleic acids on aSouthern blot. In the presence of homologous nucleic acid, ahybridization complex is produced between the nucleic acid and theoligonucleotide probe. Hybridization between nucleic acid molecules isdiscussed in detail in Sambrook et al. (1989, Molecular Cloning: ALaboratory Manual, 2^(nd) Ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.; Sections 7.37-7.57, 9.47-9.57, 11.7-11.8, and11.45-11.57).

For oligonucleotide probes less than about 100 nucleotides, Sambrook etal. discloses suitable Southern blot conditions in Sections 11.45-11.46.The Tm between a sequence that is less than 100 nucleotides in lengthand a second sequence can be calculated using the formula provided inSection 11.46. Sambrook et al. additionally discloses prehybridizationand hybridization conditions for a Southern blot that usesoligonucleotide probes greater than about 100 nucleotides (see Sections9.47-9.52). Hybridizations with an oligonucleotide greater than 100nucleotides generally are performed 15-25° C. below the Tm. The Tmbetween a sequence greater than 100 nucleotides in length and a secondsequence can be calculated using the formula provided in Sections9.50-9.51 of Sambrook et al. Additionally, Sambrook et al. recommendsthe conditions indicated in Section 9.54 for washing a Southern blotthat has been probed with an oligonucleotide greater than about 100nucleotides.

The conditions under which membranes containing nucleic acids areprehybridized and hybridized, as well as the conditions under whichmembranes containing nucleic acids are washed to remove excess andnon-specifically bound probe can play a significant role in thestringency of the hybridization. Such hybridizations can be performed,where appropriate, under moderate or high stringency conditions. Suchconditions are described, for example, in Sambrook et al. section11.45-11.46. For example, washing conditions can be made more stringentby decreasing the salt concentration in the wash solutions and/or byincreasing the temperature at which the washes are performed. Inaddition, interpreting the amount of hybridization can be affected, forexample, by the specific activity of the labeled oligonucleotide probe,by the number of probe-binding sites on the template nucleic acid towhich the probe has hybridized, and by the amount of exposure of anautoradiograph or other detection medium.

It will be readily appreciated by those of ordinary skill in the artthat although any number of hybridization and washing conditions can beused to examine hybridization of a probe nucleic acid molecule toimmobilized target nucleic acids, it is more important to examinehybridization of a probe to target nucleic acids under identicalhybridization, washing, and exposure conditions. Preferably, the targetnucleic acids are on the same membrane.

A nucleic acid molecule is deemed to hybridize to a target nucleic acidbut not to a non-homologous nucleic acid if hybridization to thehomologous target nucleic acid is at least 5-fold (e.g., at least6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 50-fold, or 100-fold)greater than hybridization to the non-homologous nucleic acid. Theamount of hybridization can be quantitated directly on a membrane orfrom an autoradiograph using, for example, a PhosphorImager or aDensitometer (Molecular Dynamics, Sunnyvale, Calif.).

Detection of an amplification product or a hybridization complex isusually accomplished using detectable labels. The term “label” withregard to a nucleic acid is intended to encompass direct labeling of anucleic acid by coupling (i.e., physically linking) a detectablesubstance to the nucleic acid, as well as indirect labeling of thenucleic acid by reactivity with another reagent that is directly labeledwith a detectable substance. Detectable substances include variousenzymes, prosthetic groups, fluorescent materials, luminescentmaterials, bioluminescent materials, and radioactive materials. Examplesof suitable enzymes include horseradish peroxidase, alkalinephosphatase, β-galactosidase, or acetylcholinesterase; examples ofsuitable prosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; examples ofbioluminescent materials include luciferase, luciferin, and aequorin,and examples of suitable radioactive material include ¹²⁵I, ¹³¹I, ³⁵S or³H. An example of indirect labeling includes end-labeling a nucleic acidwith biotin such that it can be detected with fluorescently labeledstreptavidin.

Therapeutic Uses of VEGF-C RNA Molecules

According to the methods of the invention, the expression of VEGF-C canbe reduced by introducing a VEGF-C nucleic acid molecule of theinvention into a cell. For example, the expression of VEGF-C can bereduced in a cancer cell or any other cell in which a reduction inVEGF-C is desirable. A reduction in expression of VEGF-C can be due to areduction in the amount of VEGF-C mRNA and/or the encoded polypeptide,and is reduced compared to expression in the absence of the nucleic acidmolecule. The term “reduced” is used herein to indicate that expressionof VEGF-C is reduced by 1-100% (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, 95%, or 99% reduced). “Knock-down” or “knock-down technology”refers to techniques in which the expression of a target nucleic acid isreduced compared to expression of the target nucleic acid in the absenceof a RNA molecule.

The term “VEGF-C” is meant to refer to an enzyme that is a member of thevascular endothelial growth factor family, and is involved inangiogenesis. “VEGF-C” also refers to the nucleic acid (DNA or RNA)encoding such an enzyme. Representative sequences of VEGF-C can befound, for example, without limitation, in GenBank Accession Nos.NM_(—)053653 and AY032729 (Rattus norvegicus), AF009178 (Bos taurus),NM_(—)005429 and X94216 (Homo sapiens), NM_(—)009506 and U73620 (Musmusculus), and Y15837 (Coturnix coturnix).

For example, the expression of VEGF-C mRNA can be reduced in a cell byantisense, RNAi, or siRNA. A siRNA can be two separate RNA moleculesthat hybridize together, or a single molecule that forms a hairpin.

The VEGF-C nucleic acid molecules of the invention can be used to reducethe expression of VEGF-C in a number of cell types or tissue types. Forexample, the VEGF-C nucleic acid molecules of the invention can be usedto reduce the expression of VEGF-C in cancer cells. As used herein,“cancer cells” refer to cells that grow uncontrollably and/orabnormally, and can be, for example, epithelial carcinomas. Epithelialcarcinomas include, for example, head and neck cancer cells, breastcancer cells, prostate cancer cells, and colon cancer cells. The nucleicacid molecules of the invention are preferably administered so as toresult in an inhibition of the proliferation of cancer cells.Proliferation of cancer cells as used herein refers to an increase inthe number of cancer cells (in vitro or in vivo) over a given period oftime (e.g., hours, days, weeks, or months). It is noted that the numberof cancer cells is not static and reflects both the number of cellsundergoing cell division and the number of cells dying (e.g., byapoptosis). An inhibition of the proliferation of cancer cells can bedefined as a decrease in the rate of increase in cancer cell number, acomplete loss of cancer cells, or any variation therebetween. Withrespect to tumors, a decrease in the size of a tumor can be anindication of an inhibition of proliferation.

The amount of a nucleic acid molecule administered will vary dependingon various factors including, but not limited to, the compositionchosen, the particular type and stage of cancer, the weight, thephysical condition, and the age of the individual, and whetherprevention or treatment is to be achieved. Such factors can be readilydetermined by a clinician using animal models or other test systems thatare well known in the art. The nucleic acids of the present inventioncan be delivered to a cell in a number of ways. For example, a nucleicacid molecule of the invention (e.g., a siRNA) can be directlyadministered to a cell, or a vector encoding a nucleic acid molecule ofthe invention (e.g., a viral vector) can be administered to a cell.Viral vectors include, without limitation, a lentivirus, an adenovirus,an adeno-associated virus, a retrovirus, a vaccinia virus, a herpesviruses, and a bovine papilloma virus. In addition, a nucleic acidmolecule of the invention or a vector encoding such a nucleic acid canbe encapsulated in, for example, a nanoparticle or a liposome, andadministered to a cell.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. For example, thetherapeutic agent may be introduced directly into the cancer of interestvia direct injection. Additionally, examples of routes of administrationinclude parenteral, e.g. intravenous, intradermal, subcutaneous, oral(e.g., ingestion or inhalation), transdermal (topical), transmucosal,and rectal administration. siRNA molecules of the invention can beincorporated into pharmaceutical compositions suitable foradministration. Such compositions typically comprise the nucleic acidmolecule, and a pharmaceutically acceptable carrier. As used herein,“pharmaceutically acceptable carrier” is intended to include any and allsolvents, dispersion media, coatings, antibacterial and anti-fungalagents, isotonic and absorption delaying agents, and the like,compatible with pharmaceutical administration. The use of such media andagents for pharmaceutically active substances is well known in the art.

Solutions or suspensions can include the following components: a sterilediluent such as water for injection, saline solution (e.g., phosphatebuffered saline (PBS)), fixed oils, a polyol (for example, glycerol,propylene glycol, and liquid polyetheylene glycol, and the like),glycerine, or other synthetic solvents; antibacterial and antifungalagents such as parabens, chlorobutanol, phenol, ascorbic acid,thimerosal, and the like; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. The properfluidity can be maintained, for example, by the use of a coating such aslecithin, by the maintenance of the required particle size in the caseof dispersion and by the use of surfactants. In many cases, it will bepreferable to include isotonic agents, for example, sugars, polyalcoholssuch as mannitol or sorbitol, and sodium chloride in the composition.Prolonged administration of the injectable compositions can be broughtabout by including an agent that delays absorption. Such agents include,for example, aluminum monostearate and gelatin. The parenteralpreparation can be enclosed in ampules, disposable syringes, or multipledose vials made of glass or plastic.

Oral compositions generally include an inert diluent or an ediblecarrier. Oral compositions can be liquid, or can be enclosed in gelatincapsules or compressed into tablets. Pharmaceutically compatible bindingagents, and/or adjuvant materials can be included as part of an oralcomposition. Tablets, pills, capsules, troches and the like can containany of the following ingredients, or compounds of a similar nature: abinder such as microcrystalline cellulose, gum tragacanth or gelatin; anexcipient such as starch or lactose; a disintegrating agent such asalginic acid, Primogel, or corn starch; a lubricant such as magnesiumstearate or Sterotes; a glidant such as colloidal silicon dioxide; asweetening agent such as sucrose or saccharin; or a flavoring agent suchas peppermint, methyl salicylate, or orange flavoring. Transmucosaladministration can be accomplished through the use of nasal sprays orsuppositories. For transdermal administration, the active compounds areformulated into ointments, salves, gels, or creams as generally known inthe art.

It is especially advantageous to formulate compositions in dosage unitform for ease of administration and uniformity of dosage. Dosage unitform as used herein refers to physically discrete units suited asunitary dosages for an individual to be treated; each unit containing apredetermined quantity of active compound calculated to produce thedesired therapeutic effect in association with the requiredpharmaceutical carrier. The dosage unit forms of the invention aredependent upon the amount of a compound necessary to inhibitproliferation of the cancer cells. The amount of a compound necessary toinhibit proliferation of the cancer cells can be formulated in a singledose, or can be formulated in multiple dosage units. Treatment mayrequire a one-time dose, or may require repeated doses.

Articles of Manufacture

The invention encompasses articles of manufacture (e.g., kits) thatcontain one or more nucleic acid molecules of the invention, or one ormore vectors that encode a nucleic acid molecule of the invention. Suchnucleic acid molecules are formulated for administration as describedherein, and are packaged appropriately for the intended route ofadministration. For example, a nucleic acid molecule of the invention ora vector encoding a nucleic acid molecule of the invention can becontained within a pharmaceutically acceptable carrier.

Kits of the invention also can include additional reagents (e.g.,buffers, co-factors, or enzymes). Pharmaceutical compositions of theinvention further can include instructions for administering thecomposition to an individual. The kit also can contain a control sampleor a series of control samples that can be assayed and compared to thebiological sample. Each component of the kit is usually enclosed withinan individual container and all of the various containers are within asingle package.

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

EXAMPLES Example 1 siRNA Design, Cloning, and DNA Preparation

Target identification and Oligonucleotide Design

Five VEGF-C siRNA sequences were screened for ability to reduce VEGF-CmRNA levels using quantitative RT-PCR. The five siRNA molecules weredistributed along the VEGF-C coding sequence. The following strategy wasused for designing the VEGF-C siRNA molecules.

At the NCBI website (ncbi.nlm.nih.gov on the World Wide Web), the FASTAversion of the VEGF-C sequence was selected and imported into the siRNAFinder program at Ambion's website (ambion.com/techlib/misc/siRNA_finderon the World Wide Web). The following parameters were then selected:siRNA's to end with TT; no more than 50% GC content; and no polyA orpolyT sequences (no more than 4 consecutive). The VEGF-C sequence wassubmitted for processing. The program is able to format theoligonucleotide sequences such that they are suitable for a particularpSilencer vector. The program designs oligonucleotides to include aleader sequence with the correct restriction site for the chosen vector,the sense sequence of the target sequence, a 9-nt hairpin (TTCAAGAGA),an antisense of the target sequence, RNA polymerase III terminationsequence (poly T), and a restriction site at the 3′ end for cloning.Unless otherwise indicated, the pSilencer vector designated 2.1-U6 wasused in the experiments described herein. The oligonucleotide sequencesidentified by the program were ordered from Sigma/Genosys (TheWoodlands, TX) on a 50 nM scale with standard desalting purification.Each construct for each target position contained 2 oligonucleotides (asense and an anti-sense strand) each around 63 to 64 nucleotides inlength.

Annealing the Oligonucleotide

Each oligonucleotide was diluted with QS PCR-grade water to 1 μg/μLusing the OD₂₆₀ reading reported from the manufacturer. Theoligonucleotide suspension was vortexed and centrifuged. 2 μL siRNAoligonucleotide (sense), 2 μL siRNA oligonucleotide (antisense), and 46μL annealing buffer (100 mM NaCl+50 mM HEPES (pH 7.4)) were combined andheated to 95° C. for 1 h in a PCR machine. The tubes were thencentrifuged, placed in a 37° C. water bath for 1 to 2 hours, and cooledto room temperature. Oligonucleotides were stored at −20° C.

Vector Ligation

Vector ligation was optimized for the pSilencer 2.1-U6 system (Ambion;Austin, Tex.). 5 μL of annealed siRNA oligonucleotides were diluted bythe addition of 45 μL PCR-grade water. The ligation reaction included 6μL nuclease-free water, 1 μL 10× T4 DNA ligase buffer, 1 μL pSilencer2.1 U6 Hygro/Neo vector, 1 μL (˜400 U) T4 DNA ligase (New EnglandBiolabs), and 1 μL of the diluted and annealed siRNA oligonucleotidesdescribed above. The ligation reaction was either incubated for 30minutes at 37° C. or for 3-12 hours at room temperature. The ligatedvectors were stored at −20° C.

Bacterial Transformation and Plating

Tubes of ONE SHOT® competent E. coli cells (Invitrogen) were thawed onice. Five μL of ligated siRNA vector was added to the competent cellsand mixed by gently flicking the tube. The mixture was incubated on icefor 30 minutes, heat-shocked for 45 sec at 42° C., and returnedimmediately to the ice for about five minutes. 250 μL SOC media(provided by the manufacturer with the competent cells) was added to themixture, and the mixture was shaken vigorously at 37° C. for 1 to 2 h.50 and 250 μL of SOC bacterial broth was plated onto individualLB-Amp+plates (50-100 μg/mL, pre-warmed to 37° C.) using sterilespreading techniques. The plates were incubated at 37° C. for 12-14hours.

Mini-Preps

Five mini preps were prepared for each VEGF-C siRNA construct. Three tofour ml of LB-Amp⁺ broth were added to each 15 ml tube. Individualcolonies were picked from the overnight plates using a sterile 200 μLpipette tip. The pipette tip was usually ejected directly into the tube.The tubes were shaken vigorously for 12 to 14 h at 37° C. using a Domannincubator. One to 3 ml of broth was removed and centrifuged in 1.5 mleppendorf tubes to pellet the bacteria. The remaining vials and brothwere stored at 4° C. Mini-preps were performed according to the Qiagenprotocol, except that the DNA was eluted using water and not the elutionbuffer supplied by the manufacturer.

One to 2 μL of eluted DNA resuspended in 5 to 6 μL water (final volumeof 7 μL) was submitted to the University of Iowa DNA Facility forsequencing. Sequencing reactions of the pSilencer 1.0-U6 vector and thepSilencer 2.1-U6 Hygro vector used the T7 core primer, and sequencingreactions of the pSilencer 2.1-U6 Neo vector used the M13 core primer.Intact siRNA sequence insertions were confirmed by comparing thesequence results with the oligonucleotides designed using the siRNAFinder computer program.

Maxi-Preps and Glycerol Stock Preparation

Once a correctly cloned insert and vector were identified, the remaining1-2 ml of the original overnight culture was used to inoculate 100 ml ofLB-Amp⁺ in a sterile 500 ml Erlenmeyer flask. The inoculated media wasshaken vigorously at ≧200 rpms at 37° C. for 12-14 h. A glycerol stockwas made of each vector using 1.5 ml of bacterial broth and 0.5 ml ofsterile 60% glycerol. The remaining broth was ultracentrifuged at 6000rpm for 15 minutes to pellet the bacteria, and the supernatant wasremoved. DNA was extracted using the Qiagen Filter Maxi-prep technique.The amount of DNA obtained was measured using OD₂₆₀/OD₂₈₀, and sampleswere frozen at −20° C.

Example 2 siRNA Testing and GFP Sorting

Dual-GFP transfection was used to test the effect of different siRNAconstructs on cellular mRNA and/or protein levels. The dual-GFPtransfection technique is the process of double-transfecting siRNAexpression vectors with a large excess of a GFP expression vector.Following 24-48 hours, the cells were sorted on a flow cytometer, andGFP-positive cells were collected.

Cell Culture and DNA Transfection

1.0 to 1.5×10⁶ UM-SCC-1 cells from each construct were seeded induplicate onto 100 mm tissue culture plates. Twenty-four hours later,cells had reached about 50-70% confluency. DNA was transfected usingQiagen's Effectene® kit. Toxicity was reduced with high quality and highpurity DNA. For transfections, a ratio of 1:3 or 1:4 (GFP:construct)generally was used (e.g., 1 μg of GFP vector DNA (pEGFP-N1 vector), and3-4 μg of construct DNA), however, a ratio of up to 1:7 also was usedsuccessfully. Preliminary experiments determined that a 1:8:10 ratio ofDNA: enhancer: effectene (1 μg of DNA, 8 μL of enhancer, and 10 μL ofeffectene) resulted in efficient transfection.

General transfection techniques for a single sample are described. TheDNA (3 μg construct and 1 μg GFP per sample) was diluted into 300 μL ECbuffer (provided with kit) in a 15 mL Falcon tube. Prior to beginningtransfection, media was removed from plates, the plates were washed oncewith PBS, and 7 mL of complete media (+ABX and serum) was added to eachplate. 32 TL of enhancer was added and the mixture was vortexed for 1sec and then incubated for five minutes at room temperature. 40 μL ofeffectene was added and the mixture was vortexed for 10 sec and thenincubated at room temperature for five minutes. Three mL of completemedia was added to the mixture, the tube was inverted to mix, and themedia was added dropwise to the plates. Approximately 20-30%transfection efficiency was observed with UM-SCC-1 cells.

Complexes were removed after 4-6 hours. Plates were washed one time withPBS and refreshed with complete media. The complexes were incubated for24-120 hours before sorting. Plates were checked daily for signs oftoxicity.

Sorting

Plates were trypsinized approximately one hour in advance of sorting,and duplicate samples were combined into a single tube. Cells werepelleted, and resuspended in 1 mL of PBS. Cells were filtered using a70μ filter, and the filtrate was transferred into flow tubes. A 15 mLFalcon® tube containing 2 to 3 mL of complete media was prepared foreach sample. The samples and the sorting tubes were transported to theflow lab for sorting.

Sample Preparation

The number of cells collected was noted for each sample. The number ofcells collected represents the GFP⁺ cells, which, by association, shouldcontain a large amount of siRNA vector. To measure RNA transcripts,100,000 cells or more (e.g., 250,000 cells) were collected. To measurepolypeptides, 250,000 cells or more (e.g., 500,000 cells) werecollected.

To examine the level of RNA transcripts, the collected cells werepelleted, the supernatant was removed, and the cells were resuspended in333 μL of Trizol (Gibco BRL, Carlsbad, Calif.). The resuspended cellswere stored at −80° C. for no more than 1 week before the RNA wasextracted. Taqman RT-PCR was used to assay for the level of mRNAexpression in the cells. Reduced levels of mRNA expression generallywere observed by 24 to 48 hours after sorting.

To examine the level of polypeptide, the collected cells were pelleted,the supernatant was removed, and the cells were resuspended in 1 mL PBS.The cells were again pelleted, the supernatant removed, and 40 to 50 μLof lysis buffer was added. The cells were heated to boiling for sevenminutes, and if necessary, stored at −20° C. A Bradford assay andWestern blotting can be used to assay for the level of polypeptide inthe cells. Reduced levels of polypeptides generally were observed for upto 70 to 120 hours after sorting.

Example 3 VEGF-C siRNA Sequences and Results

The VEGF-C siRNAs were designated as described in Example 1. The VEGF-CsiRNA sequences are shown in Table 1. TABLE 1 Position relative to SEQID VEDF-C sequence^(a) 5′→3′ Sequence NO: 591-611 AAGATCTGGAGGAGCAGTTAC1 689-709 AAAGGAGGCTGGCAACATAAC 2 888-908 AACCTCCATGTTGTGTCCGTC 31151-1171 AAGACCTGCCCCACCAATTAC 4 1638-1658 AAGAAGTGTGTCGTTGTGTCC 5^(a)GenBank Accession No. NM_005429

Double-transfected UM-SCC-1 cells were sorted for GFP expressionfollowed by RNA extraction and/or protein extraction and PCR or Westernblot. Each oligonucleotide sequence was screened in direct comparison toa scrambled control RNA molecule that does not target any known humangene but does initiate the RNAi cascade. FIG. 1 shows the results of arepresentative experiment with the data presented as the percent ofcontrol mRNA levels. The five VEGF-C siRNA molecules designed reducedthe accumulation of VEGF-C in cells by about 54% to about 83%.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1. An isolated nucleic acid molecule comprising a first portion, whereinthe first portion is no more than 30 nucleotides in length, wherein thefirst portion comprises 5′-AAG ATC TGG AGG AGC AGT TAC-3′ (SEQ ID NO:1),5′-AAA GGA GGC TGG CAA CAT AAC-3′ (SEQ ID NO:2), 5′-AAC CTC CAT GTT GTGTCC GTC-3′ (SEQ ID NO:3), or 5′-AAG AAG TGT GTC GTT GTG TCC-3′ (SEQ IDNO:5).
 2. The nucleic acid molecule of claim 1, further comprising asecond portion, wherein the second portion has a sequence that iscomplementary to the first portion.
 3. The nucleic acid molecule ofclaim 2, further comprising a linking sequence that joins the firstportion and the second portion.
 4. The nucleic acid molecule of claim 3,wherein the linking sequence forms a loop of a hairpin.
 5. The nucleicacid molecule of claim 4, wherein the linking sequence is about 4 toabout 10 nucleotides in length.
 6. The nucleic acid molecule of claim 1,wherein the first portion is from about 19 to about 23 nucleotides inlength.
 7. A vector comprising the nucleic acid molecule of claim
 1. 8.A host cell comprising the vector of claim
 7. 9. A method of reducingthe expression of VEGF-C in a cell, comprising the step of introducingthe isolated nucleic acid molecule of claim 1 or the vector of claim 7in to the cell in an amount sufficient to reduce the expression ofVEGF-C in the cell.
 10. The method of claim 9, wherein expression ofVEGF-C is reduced by at least 10%.
 11. The method of claim 9, whereinthe cell is a cancer cell.
 12. The method of claim 11, wherein thecancer cell is an epithelially-derived cancer cell.
 13. The method ofclaim 12, wherein the epithelially-derived cancer cell is a head andneck cancer cell, a breast cancer cell, a colon cancer cell, or aprostate cancer cell.
 14. The method of claim 11, wherein the cancercell is in vivo.
 15. The method of claim 11, wherein the cancer cell isin a mammal.
 16. The method of claim 15, wherein the mammal is a human.17. The method of claim 11, wherein proliferation of the cancer cell isinhibited.
 18. A pharmaceutical composition comprising the isolatednucleic acid molecule of claim 1 or the vector of claim 7.